Roles of cell adhosive peptides from laminins in organogenesis

1. 免疫组织化学方法研究了层粘连蛋白-5和层粘连蛋白-α5链在发育中的小鼠颌下腺组织中的分布。laminin-5在发育中的导管基底膜中表达，而上皮终芽中不表达。而在胚胎早期，层粘连蛋白-α5链在整个上皮基底膜上均有表达，提示层粘连蛋白-α5链在上皮形态发生中的作用。为了阐明层粘连蛋白α5链在器官发生中的作用，我们在颌下腺器官培养中测试了从层粘连蛋白α5链序列的羧基端球状结构域合成的15个细胞粘附肽。我们发现一个肽序列LVLFLNHGHFVA（残基3307-3318，命名为A5G-77）在250μg/ml时强烈抑制器官发育。合成了A5G-77的各种氨基端和羧基端截断肽，并在同一体系中测试了它们的活性。羧基末端缺失肽A5G-77f （LVLFLNHGH）对器官发育有抑制作用，而氨基末端缺失肽A5G-77c （FLNHGHFVA）对器官发育没有抑制作用。a5g -77处理的外植体形态与ag -73处理的外植体形态不同，说明各层粘连蛋白α链在器官发育中的作用不同。建立了无血清的胚胎13日龄小鼠离体振动萌芽器官培养体系。该系统允许我们测试22层粘胶蛋白-1肽在振动生长和分化中的作用。我们发现RKRLQVQLSIRT序列位于层粘连蛋白-α1链羧基末端g结构域的LG-4亚结构域，并指定为AG-73，可以干扰体外振动生长。其他细胞黏附层粘连蛋白-1多肽和一种对照混乱肽LQQRRSVLRTKI对振动生长没有任何显著影响。AG-73处理的振动表皮基底膜形成受损，胞质泡形成异常，肌动蛋白束基底形成异常，表明AG-73信号参与细胞骨架组织。少

1. The distributions of laminin-5 and laminin-α5 chain, were studied immunohistochemically in the developing submandibular glands of mice. The expression of laminin-5 was found in the basement membrane of developing duct but not epithelial end bud. In contrast, laminin-α5 chain was expressed in the entire epithelial basement membrane at the early embryonic stage, suggesting the role of laminin-α5 chain on the epithelial morphogenesis.2. To elucidate roles of the laminin α5 chain on organogenesis, we tested 15 cell-adhesive synthetic peptides from a carboxy-terminal globular domain of laminin-α5-chain sequence in submandibular gland organ culture. We found a peptide sequence LVLFLNHGHFVA (residues 3307-3318 and designated A5G-77) strongly inhibits the organ development at 250μg/ml. Various amino- and carboxy-terminal truncated peptides of A5G-77 were synthesized and their activities were also tested in the same system. A carboxy-treminal deletion peptide A5G-77f (LVLFLNHGH) worked inhib … More itory, whereas an amino-treminal deletion peptide A5G-77c (FLNHGHFVA) failed to inhibit the organ development. The morphology of A5G-77-treated explants differed from those of AG-73-treated, indicating distinct role of individual laminin α chains for organ development.3. We established a serum-free organ culture system of isolated vibrissal rudiments of embryonic day-13 mice. This system allowed us to test 22 laminin-1 peptides for their roles on the vibrissal growth and differentiation. We found that RKRLQVQLSIRT sequence, which is mapped at LG-4 subdomain of laminin-α1-chain carboxyl-terminal G-domain and designated AG-73, perturbed vibrissal growth in vitro. Other cell-adhesive laminin-1 peptides and a control scrambled-peptide, LQQRRSVLRTKI, failed to show any significant effects on vibrissal growth. An impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in AG-73-treated vibrissal epidermis, indicating that AG-73 signaling is involved in cytoskeletal organization. Less